Phosphoproteomic profiling reveals PI pathway and transcription factor regulation in Tamarix hispida under salt stress

Salinity is a critical abiotic stressor that adversely affects plant development and reduces yield. To explore the molecular basis of Tamarix hispida's response to salt stress, we performed an integrative analysis combining physiological measurements with proteomic and phosphoproteomic approaches. Under 200 mM NaCl treatment, physiological assays revealed that membrane damage and oxidative stress peaked at 3 h, identifying this time point as critical for capturing early stress-responsive events. Using 4D label-free quantitative proteomics, we quantified 5382 proteins, of which 509 were differentially expressed. Notably, functional enrichment analysis indicated that many suppressed proteins participated in pathways connected to photosynthesis and carbon fixation, suggesting that salt stress disrupts essential metabolic processes. Concurrently, phosphoproteomic profiling revealed 5425 phosphosites mapped to 1960 distinct proteins, including 132 differentially phosphorylated proteins (DEPPs). Functional enrichment analysis indicated that these DEPPs are primarily involved in protein phosphorylation, RNA splicing, and cell cycle regulation. Key components of the phosphatidylinositol (PI) signaling pathway-such as phosphatidylinositol-4-phosphate 5-kinase (PIP5K) and diacylglycerol kinase (DGK)-exhibited increased phosphorylation levels, implying activation of downstream messengers including inositol trisphosphate (IP3) and diacylglycerol (DAG), which may serve to amplify salt stress signaling. Additionally, enhanced phosphorylation of nuclear transport proteins, including importins and nucleoporins, suggests that nucleocytoplasmic trafficking is dynamically regulated in response to salt stress. Several transcription factors (TFs) also exhibited significant phosphorylation changes, implying their involvement in stress-responsive regulatory networks. Functional validation of five randomly selected TFs demonstrated that site-directed mutagenesis of their phosphorylation residues significantly altered salt tolerance, highlighting the pivotal role of phosphorylation in modulating transcriptional responses to salt stress. Collectively, these findings uncover a critical regulatory mechanism in T. hispida's adaptation to salinity, linking early signal transduction with transcriptional and metabolic reprogramming.